MiRNAs were measured using Taqman miRNA assay kits (Applied Biosystems, USA) according to the manufacturer's protocol. Briefly, about 30 ng enriched RNA was reverse transcribed with a TaqMan microRNA Reverse Transcription Kit (Applied Biosystems, USA) in a 15 µL reaction volume. Expression levels of miR-21 and miR-152 were quantified in triplicate by qRT-PCR using human TaqMan MicroRNA Assay Kits (Applied Biosystems, USA) on Eppendorf iplex 4 system (Eppendorf North America, Hauppauge, NY). Spiked-in Cel-miR-39 was used as a normalizer for plasma miRNA quantification.
Protocol detail
Quantitative miRNA Expression Analysis
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Corresponding Organization : Rush University Medical Center
Other organizations : Shenzhen Bao'an District People's Hospital, Fourth Affiliated Hospital of Guangxi Medical University, University of Arizona, Johns Hopkins Medicine, Johns Hopkins University
Top 5 similar protocols
Variable analysis
independent variables
- None explicitly mentioned
- Expression levels of miR-21
- Expression levels of miR-152
- Spiked-in Cel-miR-39 used as a normalizer for plasma miRNA quantification
- Positive control: None mentioned
- Negative control: None mentioned
Annotations
Based on most similar protocols
To reduce possible high intra-assay variance introduced by low abundant miRNA, a pre-amplification step using TaqMan PreAmp Master Mix (Applied Biosystems) was performed for serum RNA samples prior to miR-210 analysis according to the manufacturer's instructions (Protocol 1).
Both spiked in cel-miR-39 and endogenous miR-16 were used as normalizers for plasma miRNA quantification, while RNU6B for tissue samples (Protocol 2).
Exogenously added cel-miR-39 served as a control for normalizing the data. The comparative Cq (ΔCq) method was used to analyze the miRNA expression levels (Protocol 3).
The expression levels of each serum miRNA were normalized against that of cel-miR-39 and then calculated with the 2^-ΔΔCt method (Protocol 4).
All qPCR reactions were performed on StepOne real-time reverse transcription (RT)-PCR (Life Technologies, USA) in triplicate for each sample. The fold changes of miRNA were normalized to a reference, synthetic small RNA, cel-miR-39 (Protocol 5).
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!